Platelet protein S directly inhibits procoagulant activity on platelets and microparticles^[*]

Summary

Anticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PSdirect that was similar to or greater than that of Zn²⁺-containing pdPS and much greater than that of Zn²⁺-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticlebound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.

Keywords

Activated protein C - protein S - platelets - microparticles - anticoagulant activity

^* A preliminary report of this study was presented in abstract form at the American Society of Hematology annual meeting, December, 2010.

• References

• 1 Tans G, Rosing J, Thomassen MC, Heeb MJ. et al. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-2648.
• 2 Schwarz HP, Heeb MJ, Wencel-Drake JD. et al. Identification and quantitation of protein S in human platelets. Blood 1985; 66: 1452-1455.
• 3 Schwarz HP, Fischer M, Hopmeier P. et al. Plasma protein S deficiency in familial thrombotic disease. Blood 1984; 64: 1297-1300.
• 4 Burstyn-Cohen T, Heeb MJ, Lemke G. Lack of protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis. J Clin Invest 2009; 119: 2942-2953.
• 5 Saller F, Brisset AC, Tchaikovski SN. et al. Generation and phenotypic analysis of protein S-deficient mice. Blood 2009; 114: 2307-2314.
• 6 Heeb MJ, Marzec U, Gruber A. et al. Antithrombotic activity of protein S infused without APC in a baboon thrombosis model. Thromb Haemost 2012; 107: 690-698.
• 7 Solymoss S, Tucker MM, Tracy PB. Kinetics of inactivation of membrane-bound FVa by APC. Protein S modulates FXa protection. J Biol Chem 1988; 263: 14884-14890.
• 8 Harris KW, Esmon CT. Protein S is required for bovine platelets to support APC binding and activity. J Biol Chem 1985; 260: 2007-2010.
• 9 Lundwall A, Dackowski W, Cohen E. et al. Isolation and sequence of the cDNA for protein S, a regulator of blood coagulation. Proc Natl Acad Sci USA 1986; 83: 6716-6720.
• 10 Mitchell CA, Salem HH. Cleavage of protein S by a platelet membrane protease. J Clin Invest 1987; 79: 374-379.
• 11 Long GL, Lu D, Xie RL. et al. Human protein S cleavage and inactivation by coagulation FXa. J Biol Chem 1998; 273: 11521-11526.
• 12 Heeb MJ, Griffin JH. APC-dependent and -independent anticoagulant activities of protein S have different structural requirements. Blood Cells Mol Dis 2002; 29: 190-199.
• 13 Brinkman HJ, Mertens K, van Mourik JA. Proteolytic cleavage of protein S during the hemostatic response. J Thromb Haemost 2005; 03: 2712-2720.
• 14 Heeb MJ, Mosher D, Griffin JH. Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation. Blood 1989; 73: 455-461.
• 15 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of FV and FVa by APC. J Biol Chem 1994; 269: 31869-31880.
• 16 O’Brien LM, Mastri M, Fay PJ. Regulation of FVIIIa by APC and protein S: Inactivation of cofactor in the intrinsic FXase. Blood 2000; 95: 1714-1720.
• 17 Fernandes N, Mosnier LO, Tonnu L. et al. Zn²⁺ -containing protein S inhibits extrinsic FX-activating complex independently of TFPI. J Thromb Haemost 2010; 08: 1976-1985.
• 18 Heeb MJ, Prashun D, Griffin JH. et al. Plasma protein S contains zinc essential for efficient APC-independent anticoagulant activity and binding to FXa, but not for efficient binding to TFPI. FASEB J 2009; 23: 2244-2253.
• 19 Chattopadhyay R, Sengupta T, Majumder R. Inhibition of intrinsic Xase by protein S. Arterioscler Thromb Vasc Biol 2012; 32: 2387-2393.
• 20 Ndonwi M, Broze Jr G. Protein S enhances the TFPI inhibition of FXa but not its inhibition of FVIIa-tissue factor. J Thromb Haemost 2008; 06: 1044-1046.
• 21 Hackeng TM, Sere KM, Tans G. et al. Protein S stimulates inhibition of the tissue factor pathway by TFPI. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
• 22 Heeb MJ, Koenen RR, Fernandez JA. et al. Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals. J Thromb Haemost 2004; 02: 1766-1773.
• 23 Dahlbäk B, Wiedmer T, Sims PJ. Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles. Biochem 1992; 31: 12769-12777.
• 24 Heeb MJ, Schuck P, Xu X. Protein S multimers and monomers each have direct anticoagulant activity. J Thromb Haemost 2006; 04: 385-391.
• 25 Niemetz J, Fallon JT, Harrington E. et al. Rapid generation of thrombin by atheroma and platelets. J Thromb Haemost 2004; 02: 321-326.
• 26 Heeb MJ, Mesters R, Tans G. et al. Binding of protein S to FVa associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-2877.
• 27 Ozawa H, Kobayashi I, Marumo M. Is platelet protein S different from plasma protein S in human being?. Japan J Clin Med 1992; 50: 292-296.
• 28 Monkovic DD, Tracy PB. Functional characterization of platelet-released FV and its activation by FXa and thrombin. J Biol Chem 1990; 265: 17132-17140.
• 29 Kane WH, Mruk JS, Majerus PW. Activation of coagulation FV by a platelet protease. J Clin Invest 1982; 70: 1092-1100.
• 30 Rand MD, Kalafatis M, Mann KG. Platelet coagulation FVa: The major secretory platelet phosphoprotein. Blood 1994; 83: 2180-2190.
• 31 Gould WR, Silveira JR, Tracy PB. Unique in vivo modifications of coagulation FV produce a physically and functionally distinct platelet-derived cofactor: Characterization of purified platelet-derived FV/Va. J Biol Chem 2004; 279: 2383-2393.
• 32 Stavenuiter F, Heeb MJ. Phosphorylation of protein S by platelet kinases enhances its direct anticoagulant activity. J Thromb Haemost. 2011 Vol 9. [abstract]